Attenuated mycoplasma gallisepticum strains

ABSTRACT

The present invention provides live, attenuated  Mycoplasma gallisepticum  bacteria that exhibit reduced expression of a protein identified as MGA_0621. In certain embodiments, the attenuated bacteria may additionally exhibit reduced expression of one or more proteins selected from the group consisting of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35, relative to a wild-type  M. gallisepticum  bacterium. Also provided are vaccines and vaccination methods involving the use of the live, attenuated  M. gallisepticum  bacteria, and methods for making live attenuated  M. gallisepticum  bacteria. An exemplary live, attenuated strain of  M. gallisepticum  is provided, designated MGx+47, which was shown by proteomics analysis to exhibit significantly reduced expression of MGA_0621, and was shown to be safe and effective when administered as a vaccine against  M. gallisepticum  infection in chickens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of co-pending U.S.application Ser. No. 12/207,750, allowed, filed Sep. 10, 2008 whichclaims priority to U.S. provisional application No. 60/993,447, filedSep. 11, 2007. The entire disclosures are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of microbiology andimmunology. More specifically, the invention relates to novel vaccinesagainst bacterial pathogens.

BACKGROUND ART

Mycoplasmas are small prokaryotic organisms (0.2 to 0.3 μm) belonging tothe class Mollicutes, whose members lack a cell wall and have a smallgenome size. The mollicutes include at least 100 species of Mycoplasma.Mycoplasma species are the causative agents of several diseases in humanand non-human animals as well as in plants. M. gallisepticum, forexample, is responsible for significant disease conditions in poultry.M. gallisepticum is associated with acute respiratory disease inchickens and turkeys and can also cause upper respiratory disease ingame birds. In addition, M. gallisepticum has been recognized as a causeof conjunctivitis in house finches in North America.

An effective strategy for preventing and managing diseases caused by M.gallisepticum infection is by vaccination with live, attenuated strainsof M. gallisepticum bacteria. The advantages of live attenuatedvaccines, in general, include the presentation of all the relevantimmunogenic determinants of an infectious agent in its natural form tothe host's immune system, and the need for relatively small amounts ofthe immunizing agent due to the ability of the agent to multiply in thevaccinated host.

Live attenuated vaccine strains are often created by serially passaginga virulent strain multiple times in media. Although live attenuatedvaccine strains against M. gallisepticum have been obtained by serialpassaging, such strains are generally poorly characterized at themolecular level. It is assumed that attenuated strains made by serialpassaging have accumulated mutations which render the microorganismsless virulent but still capable of replication. With regard toattenuated M. gallisepticum strains, however, the consequences of themutations that result in attenuation (e.g., the identity of proteinswhose expression pattern has been altered in the attenuated strain) areusually unknown.

Accordingly, a need exists in the art for new live, attenuated M.gallisepticum bacteria that have been characterized at the proteomiclevel and that are safe and effective in vaccine formulations.

BRIEF SUMMARY OF THE INVENTION

The present invention is based, in part, on the surprising discoverythat M. gallisepticum bacteria that exhibit reduced expression of apolypeptide having the amino acid sequence of SEQ ID NO:1 are both safeand effective when used as a vaccine against M. gallisepticum infectionin birds. The polypeptide of SEQ ID NO:1 is also referred to as“MGA_(—)0621,” and has NCBI Accession No. NP_(—)852784.

Accordingly, the present invention is directed to live, attenuated M.gallisepticum bacteria that exhibit reduced expression of MGA_(—)0621,relative to a wild-type M. gallisepticum. In a specific, non-limiting,exemplary embodiment, the invention provides a live, attenuated M.gallisepticum strain that exhibits reduced expression of MGA_(—)0621,and further exhibits reduced expression of one or more proteins selectedfrom the group consisting of pyruvate dehydrogenase, phosphopyruvatehydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal proteinL35, relative to wild-type M. gallisepticum bacteria. According tocertain embodiments of the present invention, the live, attenuated M.gallisepticum bacteria of the invention are characterized by proteomicanalysis as having reduced expression of one or more of theaforementioned proteins. According to one exemplary embodiment of thepresent invention, the live attenuated M. gallisepticum strain is astrain that exhibits reduced expression of MGA_(—)0621, pyruvatedehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphatealdolase, and ribosomal protein L35, relative to wild-type M.gallisepticum bacteria, which strain was deposited with the AmericanType Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108, onJun. 19, 2007, and has been assigned Accession No. PTA-8485. This strainis alternatively referred to herein as “M. gallisepticum strain MGx+47,”or “MG-P48”.

The present invention also provides vaccine compositions comprising thelive, attenuated M. gallisepticum bacteria of the invention, as well asmethods of vaccinating an animal against M. gallisepticum infection.

In addition, the present invention provides methods for making and/oridentifying attenuated M. gallisepticum clones. According to this aspectof the invention, the methods comprise subjecting an initial populationof M. gallisepticum bacteria to attenuating conditions, assayingindividual clones for reduced expression of MGA_(—)0621, relative to awild-type M. gallisepticum, and testing the clones for virulence. M.gallisepticum clones produced according to the methods of this aspect ofthe invention will exhibit reduced expression of MGA_(—)0621, and mayoptionally exhibit reduced expression of one or more additional proteinsselected from the group consisting of pyruvate dehydrogenase,phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, andribosomal protein L35. Preferably, the strains that exhibit reducedexpression of at least one of the aforementioned proteins also exhibitreduced virulence relative to a wild-type M. gallisepticum bacterium.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a photograph of a two-dimensional (2-D) polyacrylamide geldepicting protein spots of the attenuated M. gallisepticum strainMGx+47. Circled spots numbered 19, 49, 74, 108, 114, 127, 147, 166, 175and 225 correspond to proteins that are up-regulated in MGx+47 relativeto wild-type strain R-980. Circled spots numbered 40, 68, 98, 99, 130,136 and 217 correspond to proteins that are down-regulated in MGx+47relative to wild-type strain R-980.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to live, attenuated M. gallisepticumbacteria that are suitable for use in vaccine formulations. The M.gallisepticum bacteria of the present invention exhibit reducedexpression of a protein referred to as MGA_(—)0621. In certainembodiments, the M. gallisepticum bacteria of the invention furtherexhibit reduced expression of one or more additional proteins selectedfrom the group consisting of pyruvate dehydrogenase, phosphopyruvatehydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal proteinL35, relative to the expression of these proteins in a wild-type M.gallisepticum bacterium of the same species.

MGA_(—)0621 is identified under NCBI Accession No. NP_(—)852784 has thefollowing 162 amino acid sequence:

(SEQ ID NO: 1) MTRTMKNKKAKKKERRFTDLSADLDEEVEKIDPEYEDFKEIKIEKNKDNQVIDKNDPFFYSESFEEARIQLIKDKKVEVKKEEEKVQETTVKNKISEAKKEEAKDVYIDSSLEIASQEPLTKGMHFYTNSRIIRKVRECAKNKGLSISRL ITMILDKSIKEE..Reduced Expression of Mycoplasma gallisepticum Proteins

A person of ordinary skill in the art will be able to determine, usingroutine molecular biological techniques, whether an attenuated M.gallisepticum bacterium exhibits reduced expression of one or moreproteins that are normally expressed in wild-type M. gallisepticumbacterial cells. Determining whether an attenuated bacterium exhibitsreduced expression of a particular protein (e.g., MGA_(—)0621, pyruvatedehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphatealdolase, ribosomal protein L35, etc.), relative to a wild-typebacterium, can be accomplished by several methods known in the art.Exemplary methods include, e.g., quantitative antibody-based methodssuch as Western blotting, radioimmunoassays (RIAs), and enzyme-linkedimmunosorbant assays (ELISAs), in which an antibody is used whichdetects and binds to the protein of interest. In addition, sincemessenger RNA (mRNA) levels generally reflect the quantity of theprotein encoded therefrom, quantitative nucleic acid-based methods mayalso be used to determine whether an attenuated M. gallisepticumbacterium exhibits reduced expression of one or more proteins. Forexample, quantitative reverse-transcriptase/polymerase chain reaction(RT-PCR) methods may be used to measure the quantity of mRNAcorresponding to a particular protein of interest. Numerous quantitativenucleic acid-based methods are well known in the art.

The following is a non-limiting, exemplary method that can be used fordetermining whether an attenuated M. gallisepticum bacterium exhibitsreduced expression of a protein such as, e.g., MGA_(—)0621.

First, a population of attenuated M. gallisepticum cells and apopulation of wild-type M. gallisepticum cells are grown undersubstantially identical conditions in substantially the same culturemedium. Next, the two populations of cells are subjected tocell-disrupting conditions. The disrupted cells (or theprotein-containing fractions thereof) are subjected, in parallel, to SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and then to Westernblotting using an antibody which binds to the M. gallisepticumMGA_(—)0621 protein (such antibodies can be obtained using standardmethods that are well known in the art). A labeled secondary antibody isthen applied in order to provide a measurable signal that isproportional to the amount of the protein derived from the cells. If theamount of signal exhibited by the attenuated M. gallisepticum strain isless than the amount of signal exhibited by the wild-type M.gallisepticum strain, then it can be concluded that the attenuatedstrain exhibits reduced expression of MGA_(—)0621 relative to thewild-type strain. Variations on this exemplary method, as well asalternatives thereto, will be immediately evident to persons of ordinaryskill in the art.

The present invention includes attenuated M. gallisepticum bacteria thatexhibit any degree of reduction in expression of a protein (e.g.,MGA_(—)0621, pyruvate dehydrogenase, phosphopyruvate hydratase,2-deoxyribose-5-phosphate aldolase, ribosomal protein L35, etc.)compared to the expression of that protein observed in a wild-typestrain. In certain embodiments, the attenuated bacterium exhibits atleast about 5% less expression of the protein relative to a wild-typebacterium. As an example, if a given quantity of a wild-type M.gallisepticum strain exhibit 100 units of expression of a particularprotein and the same quantity of a candidate attenuated M. gallisepticumstrain exhibits 95 units of expression of the protein, then it isconcluded that the attenuated strain exhibits 5% less expression of theprotein relative to the wild-type bacterium (additional examples forcalculating “percent less expression” are set forth elsewhere herein).In certain other embodiments, the attenuated bacterium exhibits at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% lessexpression of the protein relative to a wild-type M. gallisepticumbacterium. In yet other embodiments, the attenuated M. gallisepticumstrain exhibits no expression (i.e., 100% less expression) of theprotein relative to a wild-type M. gallisepticum bacterium.

In certain exemplary embodiments of the present invention, theattenuated bacteria exhibit at least 5% less expression of MGA_(—)0621,and optionally at least 5% less expression of one or more proteinsselected from the group consisting of pyruvate dehydrogenase,phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, andribosomal protein L35, relative to a wild-type M. gallisepticumbacterium.

As used herein, the “percent less expression” of a particular proteinexhibited by an attenuated M. gallisepticum strain relative to awild-type strain is calculated by the following formula: (A−B)/A×100;wherein A=the relative level of expression of the protein in a wild-typeM. gallisepticum strain; and B=the relative level of expression of theprotein in the attenuated strain. Solely for the purpose ofillustration, if a wild-type M. gallisepticum strain exhibited 0.2500units of expression of protein “Y”, and an attenuated strain of M.gallisepticum exhibited 0.1850 units of expression of protein “Y” thenthe attenuated strain is said to exhibit[(0.2500−0.1850)/0.2500×100]=26% less expression of protein “Y” relativeto the wild-type strain. Table 5 in Example 3 herein provides additionalillustrative examples of percent less expression calculated for anexemplary attenuated strain of M. gallisepticum relative to a wild-typeM. gallisepticum strain.

Vaccine Compositions

The present invention also includes vaccine compositions comprising alive, attenuated M. gallisepticum bacterium of the invention and apharmaceutically acceptable carrier. As used herein, the expression“live, attenuated M. gallisepticum bacterium of the invention”encompasses any live, attenuated M. gallisepticum bacterium that isdescribed and/or claimed elsewhere herein. The pharmaceuticallyacceptable carrier can be, e.g., water, a stabilizer, a preservative,culture medium, or a buffer. Vaccine formulations comprising theattenuated M. gallisepticum bacteria of the invention can be prepared inthe form of a suspension or in a lyophilized form or, alternatively, ina frozen form. If frozen, glycerol or other similar agents may be addedto enhance stability when frozen.

Methods of Vaccinating an Animal

The present invention also includes methods of vaccinating an animalagainst M. gallisepticum infection. The methods according to this aspectof the invention comprise administering to an animal animmunologically-effective amount of a vaccine composition comprising alive, attenuated M. gallisepticum bacterium of the invention. As usedherein, the expression “live, attenuated M. gallisepticum bacterium ofthe invention” encompasses any live, attenuated M. gallisepticumbacterium that is described and/or claimed elsewhere herein. Theexpression “immunologically-effective amount” means that amount ofvaccine composition required to invoke the production of protectivelevels of antibodies in an animal upon vaccination. The vaccinecomposition may be administered to the animal in any manner known in theart including oral, intranasal, mucosal, topical, transdermal, andparenteral (e.g., intravenous, intraperitoneal, intradermal,subcutaneous or intramuscular) routes. Administration can also beachieved using needle-free delivery devices. Administration can beachieved using a combination of routes, e.g., first administration usinga parental route and subsequent administration using a mucosal route,etc.

The animal to which the attenuated M. gallisepticum strain isadministered is preferably a bird, e.g., a chicken or a turkey. Wherethe animal is a bird, the vaccine formulations of the invention may beadministered such that the formulations are immediately or eventuallybrought into contact with the bird's respiratory mucosal membranes.Thus, the vaccine formulations may be administered to birds, e.g.,intranasally, orally, and/or intraocularly. The vaccine compositions foravian administration may be formulated as described above and/or in aform suitable for administration by spray, including aerosol (forintranasal administration) or in drinking water (for oraladministration).

Vaccine compositions of the present invention that are administered byspray or aerosol can be formulated by incorporating the live, attenuatedM. gallisepticum bacteria into small liquid particles. The particles canhave an initial droplet size of between about 10 μm to about 100 μm.Such particles can be generated by, e.g., conventional spray apparatusand aerosol generators, including commercially available spraygenerators for knapsack spray, hatchery spray and atomist spray.

Methods for Making Attenuated M. gallisepticum Clones

In another aspect of the present invention, the invention providesmethods for identifying and/or making attenuated M. gallisepticumclones. The methods according to this aspect of the invention comprisesubjecting an initial population of M. gallisepticum bacteria toattenuating conditions, thereby producing a putatively attenuatedbacterial population. Next, individual clones of the putativelyattenuated bacterial population are assayed for reduced expression ofMGA_(—)0621, relative to a wild-type M. gallisepticum bacterium. Theclones that are identified as having reduced expression of MGA_(—)0621are then tested for virulence. Clones that exhibit both reducedexpression of MGA_(—)0621 and reduced virulence relative to a wild-typeM. gallisepticum bacterium are identified as attenuated M. gallisepticumclones.

According to this aspect of the invention, the “initial population of M.gallisepticum bacteria” can be any quantity of M. gallisepticumbacteria. The bacteria, in certain embodiments are wild-type bacteria.Alternatively, the bacteria may contain one or more mutations.Preferably, however, the bacteria in the initial population are clonallyidentical or substantially clonally identical; that is, the bacteriapreferably are all derived from a single parental M. gallisepticumbacterial cell and/or have identical or substantially identicalgenotypic and/or phenotypic characteristics.

As used herein, the term “attenuating conditions” means any condition orcombination of conditions which has/have the potential for introducingone or more genetic changes (e.g., nucleotide mutations) into the genomeof a M. gallisepticum bacterium. Exemplary, non-limiting, attenuatingconditions include, e.g., passaging bacteria in culture, transformingbacteria with a genome-insertable genetic element such as a transposon(e.g., a transposon that randomly inserts into the M. gallisepticumgenome), exposing bacteria to one or more mutagens (e.g., chemicalmutagens or ultraviolet light), etc. When bacterial cells are attenuatedby passaging in vitro, the cells may be passaged any number of times,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, or more times in vitro.

The initial population of M. gallisepticum cells, after being subjectedto attenuating conditions, are referred to herein as a putativelyattenuated bacterial population. Individual clones of the putativelyattenuated bacterial population can be obtained by standardmicrobiological techniques including, e.g., serially diluting the cellsand plating out individual cells on appropriate media. Once obtained,the individual clones of the putatively attenuated bacterial populationare assayed for reduced expression of MGA_(—)0621 and/or one or moreadditional specified proteins. Methods for determining whether anattenuated M. gallisepticum bacterium exhibits reduced expression of oneor more proteins that are normally expressed in wild-type M.gallisepticum bacterial cells are described elsewhere herein. Exemplarymethods include, e.g., RT-PCR-based methods, Western blot, etc.

Individual clones that are identified as having reduced expression ofMGA_(—)0621 can be tested for virulence by administration of the clonesto an animal that is susceptible to infection by the wild-type(unattenuated) version of the bacterium. As used herein, “an animal thatis susceptible to infection by a wild-type M. gallisepticum bacterium”is an animal that shows at least one clinical symptom after beingchallenged with a wild-type M. gallisepticum bacterium. Such symptomsare known to persons of ordinary skill in the art. For example, in thecase of a putatively attenuated M. gallisepticum strain that exhibitsreduced expression of, e.g., MGA_(—)0621, the strain can be administeredto, e.g., turkeys or chickens (which are normally susceptible toinfection by wild-type M. gallisepticum). Clinical symptoms of M.gallispeticum infection of poultry animals include, e.g., acuterespiratory symptoms, pericarditis, perihepatitis, air sacculitis,trachea thickening, reduced weight gain, deciliation, abnormal gobletcells, capillary distension, increased numbers of lymphocytes, plasmacells and/or heterophils, and in some cases reduced egg production.Thus, if the putatively attenuated M. gallisepticum strain, whenadministered to a chicken or turkey, results in fewer and/or less severesymptoms as compared to a turkey or chicken that has been infected witha wild-type M. gallisepticum strain, then the putatively attenuated M.gallisepticum strain is deemed to have “reduced virulence.” Any degreeof reduction in symptoms will identify the putatively attenuated strainas having reduced virulence. In certain embodiments, the putativelyattenuated strain will be avirulent.

According to the present invention, an M. gallisepticum clone thatexhibits reduced expression of MGA_(—)0621 (and/or one or moreadditional specified proteins), and that exhibits reduced virulencerelative to a wild-type M. gallisepticum bacterium is an attenuated M.gallisepticum clone. An exemplary, live, attenuated M. gallisepticumclone of the present invention, which exhibits reduced expression ofMGA_(—)0621 (along with reduced expression of pyruvate dehydrogenase,phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, andribosomal protein L35) is the strain designated MGx+47. MGx+47 has beendeposited with the American Type Culture Collection, P.O. Box 1549,Manassas, Va. 20108, on Jun. 19, 2007 and was assigned accession numberPTA-8485.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in molecular biology and chemistry which are obvious tothose skilled in the art in view of the present disclosure are withinthe spirit and scope of the invention.

EXAMPLES Example 1 Generation of a Live, Attenuated M. gallisepticumStrain

A new live, attenuated Mycoplasma gallisepticum strain was generated bypassaging a wild-type M. galliespticum strain R980 multiple times invitro. In particular, 0.1 mL seed material of wild-type M. gallisepticumstrain R-980 was inoculated into 20 mL of modified Frey's medium (Freyet al., Am. J. Vet. Res. 29:2163-2171 (1968) (also referred to herein as“MG culture medium”). The wild-type cells were grown until media colorchanged to bright yellow. The bright yellow cultures were subsequentlyused to re-inoculate fresh MG culture media as described above. Theculture was passaged a total of 47 times in this manner. The resultingstrain was tested for attenuation by vaccinating groups of birdsfollowed by challenge using the wild-type M. gallisepticum. All thebirds were necropsized two weeks post-challenge and mycoplasma relatedpathologies were observed. High passage strain (x+47) providedprotection against the clinical signs associated with Mycoplasmagallisepticum infection. This attenuated M. gallisepticum straindesignated MGx+47 was deposited with the American Type CultureCollection, P.O. Box 1549, Manassas, Va. 20108, on Jun. 19, 2007 and wasassigned accession number PTA-8485.

Example 2 Safety and Efficacy Evaluation of a Live, Attenuated M.gallisepticum Vaccine in Chickens

In this Example, the safety and efficacy of the new M. gallisepticumvaccine strain MGx+47 obtained in Example 1 was assessed in chickens.

Seventy one SPF white leghorn chickens were divided into seven groups asfollows:

TABLE 1 Study Design Group # Chickens Vaccinated Challenged 1 11 No Yes2 10 Yes No 3 11 Yes Yes 4a 10 Yes No 4b 11 Yes No 4c 9 Yes No 5 9 No No

The chickens in groups 2, 3, 4a, 4b and 4c were vaccinated withattenuated strain MGx+47 at 3.62×10⁷ CCU/mL/bird, administered by coarsespray at 4 weeks of age. The chickens in groups 1 and 3 were challengedintratracheally (IT) at 7 weeks of age with 0.5 mL of Mycoplasmagallisepticum strain R at 7.74×10⁵ CCU/mL. Necropsy was performed on thechickens of groups 1, 2, 3 and 5 at 9 weeks of age, and necropsy wasperformed on the chickens of groups 4a, 4b and 4c at 7, 14 and 21 dayspost vaccination (DPV), respectively. The chickens were assessed foraverage weight gain, pericarditis, perihepatitis, airsacculitis, andtracheitis. The results are summarized in Table 2.

TABLE 2 Safety and Efficacy Summary Average Airsacculitis Weight GainScore (average Trachea Group Vaccinated Challenged (kg/day) PericarditisPerihepatitis Airsacculitis of positives) (Histology) 1 No Yes 0.0160/11 0/11 9/11 3.56 severe tracheitis 2 Yes No 0.018 0/10 0/10 0/10 0normal 3 Yes Yes 0.017 0/11 0/11 2/11 2.5 mixed tracheitis  4a Yes No0.016 0/9  0/9  0/9  0 normal  4b Yes No 0.017 0/11 0/11 0/11 0 normal 4c Yes No 0.017 0/10 0/10 0/10 0 normal 5 No No 0.015 0/9  0/9  0/9  0normal Vaccination = 3.62 × 10⁷ CFU/mL/bird Challenge = 0.5 mL at 7.74 ×10⁵ CFU/mL

TABLE 3 Safety Table: Histology Report of Formalin-Fixed ChickenTracheas from Individual Vaccinated/Unchallenged Chickens (Group 4a, 4band 4c) Capil- lary Thick- Time Chick- Goblet Disten- LC/ ness Point enCilia Cells/M sion PC PMNs (microns)  7 DPV 1 N − − − − 30 2 N − − − −30 3 N − − − − 30 4 N − − + − 30 5 N − − − − 30 6 N − − + − 30 7 N − − +− 30 8 N − − − − 30 9 N + − − − 30 14 DPV 1 N − − − − 50 2 N + − − − 503 N − − + − 50 4 N − − − − 50 5 N − − − − 50 6 N − − − − 50 7 N − − − −50 8 N − − − − 50 9 N − − + − 50 10 N − − − − 50 11 N − − + − 50 21 DPV1 N − − − − 50 2 N − − ++ − 110 3 N − − − − 50 4 N − − − − 50 5 N − − −− 50 6 N − − + − 50 7 N − − − − 50 8 N − − − − 50 9 N − − − − 50 10 N −− − − 50

TABLE 4 Efficacy Table: Histology Report of Formalin-Fixed ChickenTracheas from Individual Chickens Capil- lary Thick- Chick- GobletDisten- LC/ ness Group en Cilia Cells/M sion PC PMNs (microns) 1 NotVaccinated; Challenged 1 − + ++ ++++ ++ 410 2 +/− − − + − 90 3 N + − − −50 4 − − ++++ ++++ − 420 5 N + + + − 60 6 − + ++++ ++++ +++ 400 7 − −++++ ++++ − 440 8 − − ++++ ++++ ++++ 280 9 − + − − − 40 10 − − ++++ ++++− 260 11 − + ++++ ++++ +++ 450 3 Vaccinated and Challenged 1 − − ++ ++++− 380 2 N − + + − 40 3 N − + + − 50 4 − − + +++ ++ 220 5 N − + + − 60 6N − + + − 60 7 N − − − − 50 8 N − − − − 50 9 N − + + − 50 10 +/− − + ++− 140 5 Not Vaccinated; Not Challenged 1 N − − + − 50 2 N − − + − 50 3 N− − − − 50 4 N − − + − 50 5 N − − − − 50 6 N − − + − 50 7 N − − − − 50 8N − − + − 50 9 N − − − − 50

Key to Safety and Efficacy Tables (Tables 3 and 4):

-   -   All “vaccinated” birds were vaccinated by coarse spray with        vaccine strain MGx+47 at 3.62×10⁷ CCU/mL/bird;    -   All “challenged” birds were challenged intratracheally (IT) with        0.5 mL of Mycoplasma gallisepticum strain R at 7.74×10⁵ CCU/mL    -   Time Point (in Table 3: Safety Table)=number of days after        vaccination when the chickens were examined, expressed as # days        post vaccination (DPV).    -   Cilia: “N”=normal cilia; “−”=deciliation;    -   Goblet Cells/M (“−”=normal goblet cells; “+”=mucus lying on the        respiratory surface);    -   Capillary Distension (“−”=no distension or inflammation;        “+”=moderate capillary distension or inflammation; “++”=severe        capillary distension or inflammation);    -   LC/PC=Lymphocytes and Plasma cells (“−”=none; “+”=few;        “++++”=numerous);    -   PMNs=Heterophils (“−”=none; “+”=few; “++++”=numerous);

The histology analysis of the group 2 chickens (vaccinated but notchallenged) was substantially similar to that of the group 5 chickens(unvaccinated, unchallenged), demonstrating the safety of the newlygenerated MGx+47 vaccine strain. (See, e.g., Table 2 above).

With regard to efficacy, the group 3 chickens (vaccinated andchallenged) showed significantly reduced airsacculitis compared to thegroup 1 chickens (unvaccinated and challenged). (See, e.g., Tables 2 and4). In addition, as illustrated in Table 4, the group 3 chickensexhibited fewer histological signs of M. gallisepticum infection withregard to cillia, goblet cells, capillary distension, lymphocytes andplasma cells (LC/PC), heterophils (PMNs) and trachea thickness. (SeeTable 4).

Thus, this Example demonstrates that MGx+47 is a safe and effectivelive, attenuated M. gallisepticum vaccine strain.

Example 3 Proteomic Characterization of MGx+47 Vaccine Strain

In an effort to more precisely define the MGx+47 vaccine strain (seeExamples 1 and 2) at the molecular level, a proteomic analysis of thisstrain was undertaken.

In this Example, total protein was isolated from the wild-type M.gallisepticum strain R-980 and from the newly identified vaccine strainMGx+47. Proteins from each strain were resolved by 2-dimensionalpolyacrylamide gel electrophoresis followed by computerized analysis ofthe gel images. (See FIG. 1). Protein spots were identified that weredifferentially expressed in the vaccine strain. Protein spots that wereabsent, or were expressed at significantly reduced levels, in thevaccine strain compared to the wild-type strain were excised from thegel.

Five spots were identified that were expressed at significantly lowerlevels in the MGx+47 vaccine strain as compared to the wild-type M.gallisepticum. Each of these protein spots were excised from the gel andenzymatically digested. Followed by peptide mass fingerprinting usingmatrix-assisted laser desorption/ionization-time of flight massspectrometry (MALDI-TOF MS). The mass spectra identified for eachprotein spot was compared to a peptide mass database to identify theproteins and the corresponding genes that encodes them. The results ofthis analysis are summarized in the Table below:

TABLE 5 Summary of Proteomic Analysis of MGx + 47 Level of Level ofPercent expression in expression decrease in Gene Product Functionwild-type MG in MGx + 47 expression acoA Pyruvate Required for energy0.1872 0.0858 54.2% dehydrogenase production and conversion (Kreb'sCycle) eno Phospho-pyruvate Catalyzes the 0.0683 0.0173 74.7% hydrataseformation of phosphoenol- pyruvate deoC 2-deoxyribose-5- Required for0.0525 0.0309 41.1% phosphate nucleotide aldolase metabolism rpmlRibosomal protein Translaction, 0.1171 0.0259 77.9% L35 ribosomalstructure and biogenesis MGA_0621 Hypothetical Unknown 0.4534 0.083581.6% protein

The decrease in expression of the gene products can also be expressed interms of “fold decrease in expression.” For example, in Table 5, strainMGx+47 can be said to exhibit 2.2, 3.9, 1.7, 4.5 and 5.4 fold decreasedexpression of acoA, eno, deoC, rpml, and MGA_(—)0621, respectively,relative to wild-type MG.

As indicated in Table 5, five gene products were identified that hadsignificantly reduced expression in the live, attenuated MGx+47 vaccinestrain as compared to the wild-type R-980 strain: AcoA, Eno, DeoC, Rmpl,and MGA_(—)0621 (a hypothetical protein identified under NCBI accessionnumber NP_(—)852784). The largest decrease in expression was observedfor MGA_(—)0621. Thus, mutations or growth conditions which cause adecrease in MGA_(—)0621 expression are likely to result in attenuationof M. gallisepticum. Down regulation of MGA_(—)0621, therefore, appearsto be a effective strategy for producing attenuated strains of M.gallisepticum.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, this invention is not limited to the particularembodiments disclosed, but is intended to cover all changes andmodifications that are within the spirit and scope of the invention asdefined by the appended claims.

All publications and patents mentioned in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains. All publications and patents are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

1. A live, attenuated Mycoplasma gallisepticum bacterium that exhibitsreduced expression of a protein having the amino acid sequence of SEQ IDNO:1, relative to a wild-type Mycoplasma gallisepticum.
 2. The bacteriumof claim 1, wherein said bacterium exhibits at least 25% less expressionof said protein relative to a wild-type Mycoplasma gallisepticum.
 3. Thebacterium of claim 2, wherein said bacterium exhibits at least 50% lessexpression of said protein relative to a wild-type Mycoplasmagallisepticum.
 4. The bacterium of claim 3, wherein said bacteriumexhibits at least 75% less expression of said protein relative to awild-type Mycoplasma gallisepticum.
 5. The bacterium of claim 1, whereinsaid bacterium further exhibits reduced expression of pyruvatedehydrogenase relative to a wild-type Mycoplasma gallisepticum.
 6. Thebacterium of claim 1, wherein said bacterium further exhibits reducedexpression of phosphopyruvate hydratase relative to a wild-typeMycoplasma gallisepticum.
 7. The bacterium of claim 1, wherein saidbacterium further exhibits reduced expression of2-deoxyribose-5-phosphate aldolase relative to a wild-type Mycoplasmagallisepticum.
 8. The bacterium of claim 1, wherein said bacteriumfurther exhibits reduced expression of ribosomal protein L35 relative toa wild-type Mycoplasma gallisepticum.
 9. The bacterium of claim 1,wherein said bacterium further exhibits reduced expression of pyruvatedehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphatealdolase, and ribosomal protein L35 relative to a wild-type Mycoplasmagallisepticum.
 10. A vaccine composition comprising: (a) a live,attenuated Mycoplasma gallisepticum bacterium that exhibits reducedexpression of a protein having the amino acid sequence of SEQ ID NO:1,relative to a wild-type Mycoplasma gallisepticum; and (b) apharmaceutically acceptable carrier.
 11. The vaccine composition ofclaim 10, wherein said bacterium exhibits at least 25% less expressionof said protein relative to a wild-type Mycoplasma gallisepticum. 12.The vaccine composition of claim 11, wherein said bacterium exhibits atleast 50% less expression of said protein relative to a wild-typeMycoplasma gallisepticum.
 13. The vaccine composition of claim 12,wherein said bacterium exhibits at least 75% less expression of saidprotein relative to a wild-type Mycoplasma gallisepticum.
 14. Thevaccine composition of claim 10, wherein said bacterium further exhibitsreduced expression of pyruvate dehydrogenase relative to a wild-typeMycoplasma gallisepticum.
 15. The vaccine composition of claim 10,wherein said bacterium further exhibits reduced expression ofphosphopyruvate hydratase relative to a wild-type Mycoplasmagallisepticum.
 16. The vaccine composition of claim 10, wherein saidbacterium further exhibits reduced expression of2-deoxyribose-5-phosphate aldolase relative to a wild-type Mycoplasmagallisepticum.
 17. The vaccine composition of claim 10, wherein saidbacterium further exhibits reduced expression of ribosomal protein L35relative to a wild-type Mycoplasma gallisepticum.
 18. The vaccinecomposition of claim 10, wherein said bacterium further exhibits reducedexpression of pyruvate dehydrogenase, phosphopyruvate hydratase,2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35 relativeto a wild-type Mycoplasma gallisepticum.
 19. A method of vaccinating ananimal against Mycoplasma gallisepticum infection, said methodcomprising administering to an animal an immunologically-effectiveamount of a vaccine composition, said vaccine composition comprising alive, attenuated Mycoplasma gallisepticum bacterium having reducedexpression of a protein having the amino acid sequence of SEQ ID NO:1,relative to a wild-type Mycoplasma gallisepticum.
 20. The method ofclaim 19, wherein said bacterium exhibits at least 25% less expressionof said protein relative to a wild-type Mycoplasma gallisepticum. 21.The method of claim 20, wherein said bacterium exhibits at least 50%less expression of said protein relative to a wild-type Mycoplasmagallisepticum.
 22. The method of claim 21, wherein said bacteriumexhibits at least 75% less expression of said protein relative to awild-type Mycoplasma gallisepticum.
 23. The method of claim 19, whereinsaid bacterium further exhibits reduced expression of pyruvatedehydrogenase relative to a wild-type Mycoplasma gallisepticum.
 24. Themethod of claim 19, wherein said bacterium further exhibits reducedexpression of phosphopyruvate hydratase relative to a wild-typeMycoplasma gallisepticum.
 25. The method of claim 19, wherein saidbacterium further exhibits reduced expression of2-deoxyribose-5-phosphate aldolase relative to a wild-type Mycoplasmagallisepticum.
 26. The method of claim 19, wherein said bacteriumfurther exhibits reduced expression of ribosomal protein L35 relative toa wild-type Mycoplasma gallisepticum.
 27. The method of claim 19,wherein said bacterium further exhibits reduced expression of pyruvatedehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphatealdolase, and ribosomal protein L35 relative to a wild-type Mycoplasmagallisepticum.
 28. A method for identifying attenuated Mycoplasmagallisepticum clones, said method comprising: (a) subjecting an initialpopulation of Mycoplasma gallisepticum bacteria to attenuatingconditions, thereby producing a putatively attenuated bacterialpopulation; and (b) assaying individual clones of said putativelyattenuated bacterial population for reduced expression of a proteinhaving the amino acid sequence of SEQ ID NO:1, relative to a wild-typeMycoplasma gallisepticum; and (c) testing clones identified in (b) ashaving reduced expression of said protein for virulence; wherein aMycoplasma gallisepticum clone that exhibits reduced expression of saidprotein and reduced virulence relative to a wild-type Mycoplasmagallisepticum is an attenuated Mycoplasma gallisepticum clone.
 29. Themethod of claim 28, wherein said attenuating conditions of (a) comprisepassaging said initial population of Mycoplasma gallisepticum bacteriaat least 2 times in vitro.
 30. The method of claim 29, wherein saidattenuating conditions of (a) comprise passaging said initial populationof Mycoplasma gallisepticum bacteria at least 5 times in vitro.
 31. Themethod of claim 30, wherein said attenuating conditions of (a) comprisepassaging said initial population of Mycoplasma gallisepticum bacteriaat least 10 times in vitro.
 32. The method of claim 28, wherein saidattenuating conditions of (a) comprise transforming said initialpopulation of Mycoplasma gallisepticum with a transposon which randomlyinserts into the Mycoplasma gallisepticum genome.
 33. The method ofclaim 28, wherein said attenuating conditions of (a) comprise exposingsaid initial population of Mycoplasma gallisepticum to a chemicalmutagen or ultra violet light.
 34. The method of claim 28, wherein saidindividual clones of said putatively attenuated bacterial population areassayed in (b) for reduced expression of said protein by reversetranscriptase-polymerase chain reaction (RT-PCR).
 35. The method ofclaim 28, wherein said individual clones of said putatively attenuatedbacterial population are assayed in (b) for reduced expression of saidprotein by Western blot.
 36. The method of claim 28, wherein said clonesidentified in (b) are tested for virulence in (c) by administering oneor more of said clones to an animal that is susceptible to Mycoplasmagallisepticum infection and comparing the clinical symptoms observed insaid animals after being administered said one or more clones to theclinical symptoms of control animals that are not administered saidclones.
 37. The live, attenuated Mycoplasma gallisepticum bacteriumdeposited at the ATCC under accession number PTA-8485.
 38. The method ofclaim 19, wherein said vaccine composition is administered to saidanimal by direct injection, spray administration or drinking wateradministration.